Integrated Analysis of Patterning, Morphogenesis, and Cell Divisions in Embryonic Development by in toto Imaging and Quantitative Cell Tracking

Patterning, morphogenesis, and cell divisions are distinct processes during development yet are concurrent and likely highly integrated. However, it has been challenging to investigate them as a whole. Recent advances in imaging and labeling tools make it possible to observe live tissues with high coverage and resolution. In this dissertation work, we developed a novel imaging platform that allowed us to fully capture the early neural tube formation process in live zebrafish embryos at cellular resolution. Importantly, these datasets allow us to reliably track single neural progenitors. These tracks carry information on the history of cell movement, shape change, division, and gene expression all together. By comparing tracks of different progenitor fates, we found they show a spatially noisy response to Sonic hedgehog (Shh) and become specified in a positionally mixed manner, in surprising contrast to the "French Flag" morphogen patterning model. Both cell movement and division contribute to cell mixing. In addition, we decoupled the temporal and genetic regulatory network (GRN) noises in Shh interpretation using tracks that carry both Shh signaling and cell fate reporters. Our tracks suggest that, after specification, progenitors undergo sorting to self-assemble a sharp pattern. Consistent with this hypothesis, we found ectopically induced progenitors move to correct locations. Furthermore, we show that proper adhesion is required for cell sorting to happen (Chapters 2 and 3). In the cleavage stage embryos, the cells on the surface undergo shape changes followed by lineage separation and differentiation. We quantitatively measured this morphogenesis process and tracked cell divisions. By applying a mathematical model we uncover a predictive, and perhaps general link between cell division orientation, mechanical interaction, and the morphogenetic behavior of the whole surface layer (Chapter 4). Finally, we discuss the concepts and tools of cell tracking including a multi-color cell labeling method we developed by modifying the "Brainbow" system (Chapter 5). Together this dissertation showcases the importance and promise of live observation based, quantitative and integrated analysis in our understanding of complex multi-cellular developmental processes

Mechanochemical Regulation of Epithelial Tissue Remodeling: A Multiscale Computational Model of the Epithelial-Mesenchymal Transition Program

Epithelial-mesenchymal transition (EMT) regulates the cellular processes of migration, growth, and proliferation - as well as the collective cellular process of tissue remodeling - in response to mechanical and chemical stimuli in the cellular microenvironment. Cells of the epithelium form cell-cell junctions with adjacent cells to function as a barrier between the body and its environment. By distributing localized stress throughout the tissue, this mechanical coupling between cells maintains tensional homeostasis in epithelial tissue structures and provides positional information for regulating cellular processes. Whereas in vitro and in vivo models fail to capture the complex interconnectedness of EMT-associated signaling networks, previous computational models have succinctly reproduced components of the EMT program. In this work, we have developed a computational framework to evaluate the mechanochemical signaling dynamics of EMT at the molecular, cellular, and tissue scale. First, we established a model of cell-matrix and cell-cell feedback for predicting mechanical force distributions within an epithelial monolayer. These findings suggest that tensional homeostasis is the result of cytoskeletal stress distribution across cell-cell junctions, which organizes otherwise migratory cells into a stable epithelial monolayer. However, differences in phenotype-specific cell characteristics led to discrepancies in the experimental and computational observations. To better understand the role of mechanical cell-cell feedback in regulating EMT-dependent cellular processes, we introduce an EMT gene regulatory network of key epithelial and mesenchymal markers, E-cadherin and N-cadherin, coupled to a mechanically-sensitive intracellular signaling cascade. Together these signaling networks integrate mechanical cell-cell feedback with EMT-associated gene regulation. Using this approach, we demonstrate that the phenotype-specific properties collectively account for discrepancies in the computational and experimental observations. Additionally, mechanical cell-cell feedback suppresses the EMT program, which is reflected in the gene expression of the heterogeneous cell population. Together, these findings advance our understanding of the complex interplay in cell-cell and cell-matrix feedback during EMT of both normal physiological processes as well as disease progression

Constitutive Relationships and Models in Continuum Theories of Multiphase Flows

In April, 1989, a workshop on constitutive relationships and models in continuum theories of multiphase flows was held at NASA's Marshall Space Flight Center. Topics of constitutive relationships for the partial or per phase stresses, including the concept of solid phase pressure are discussed. Models used for the exchange of mass, momentum, and energy between the phases in a multiphase flow are also discussed. The program, abstracts, and texts of the presentations from the workshop are included

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Information processing in biology

To survive, organisms must respond appropriately to a variety of challenges posed by a dynamic and uncertain environment. The mechanisms underlying such responses can in general be framed as input-output devices which map environment states (inputs) to associated responses (output. In this light, it is appealing to attempt to model these systems using information theory, a well developed mathematical framework to describe input-output systems. Under the information theoretical perspective, an organism’s behavior is fully characterized by the repertoire of its outputs under different environmental conditions. Due to natural selection, it is reasonable to assume this input-output mapping has been fine tuned in such a way as to maximize the organism’s fitness. If that is the case, it should be possible to abstract away the mechanistic implementation details and obtain the general principles that lead to fitness under a certain environment. These can then be used inferentially to both generate hypotheses about the underlying implementation as well as predict novel responses under external perturbations. In this work I use information theory to address the question of how biological systems generate complex outputs using relatively simple mechanisms in a robust manner. In particular, I will examine how communication and distributed processing can lead to emergent phenomena which allow collective systems to respond in a much richer way than a single organism could

Modelling the behaviour of granular material on the surface of asteroids and under different gravity conditions (e.g., Mars, the Moon)

This thesis, at the interface between the scientific disciplines of planetary science and granular physics, has two key components, both of which intend to increase our understanding of granular dynamics in varying gravitational conditions. The dynamics of granular materials are involved in the evolution of solid planets and small bodies in our Solar System, whose surfaces are generally covered with regolith. Understanding granular dynamics is also critical for the design and/or operations of landers, sampling devices and rovers to be included in space missions. The first component of this thesis is the validation of the hard-sphere discrete element method implementation in the N-body code pkdgrav to model the dynamics of granular material. By direct comparison with results from laboratory experiments, it is demonstrated that the hard-sphere discrete element method implementation in pkdgrav is valid for modelling granular material in dilute regimes and is capable of reproducing the complex dynamical behaviour of a specific dense system as well. The second component is focussed on the AstEx parabolic flight experiment. This experiment, with the aim of characterising the response of granular material to rotational shear forces in a microgravity environment, was designed, constructed, flown and the data were analysed as part of this thesis. It was found that the effect of constant shearing on a granular material in a direction perpendicular to the gravity field is not strongly influenced by gravity. The AstEx experiment has demonstrated, for the first time, that the efficiency of granular convection may decrease in the presence of a weak gravitational field, similar to that on the surface of small bodies. The first measurements of transient weakening of granular material after shear reversal in microgravity are also reported. Results suggest that the force contact network may be weaker in microgravity, although the influence of any change in the contact network is felt by the granular material over much larger distances. This may have important implications for our interpretation of asteroid surfaces. Continued advancement of our understanding of granular materials in varying gravitational conditions requires futher experiments and the development of the soft-sphere discrete element method implementation in pkdgrav in order to model the granular regimes that are inaccessbile to the hard-sphere implementation

Natural Computing and Beyond

This book contains the joint proceedings of the Winter School of Hakodate (WSH) 2011 held in Hakodate, Japan, March 15–16, 2011, and the 6th International Workshop on Natural Computing (6th IWNC) held in Tokyo, Japan, March 28–30, 2012, organized by the Special Interest Group of Natural Computing (SIG-NAC), the Japanese Society for Artificial Intelligence (JSAI). This volume compiles refereed contributions to various aspects of natural computing, ranging from computing with slime mold, artificial chemistry, eco-physics, and synthetic biology, to computational aesthetics

INVESTIGATION INTO THE ROLES OF HER9 AND CAPN5 DURING RETINAL DEVELOPMENT AND REGENERATION

The formation of a healthy and functioning eye requires coordinated interactions between numerous signaling pathways and gene regulatory networks within the developing neural retina. Tight regulation of gene expression is required for cell specification and differentiation in this multilayered, light sensitive tissue. The photoreceptors are the light detecting cells of the retina, capable of functioning in both intense sunlight and dim light at night. When pigment cells of the photoreceptor outer segment are activated by light, a complex chain of events called phototransduction leads to the electrical signal cascade that is transmitted through the retina and ultimately to the brain to be interpreted as a visual image. Defects in expression of the genes involved in retinal and photoreceptor development have been implicated in many eye diseases, often leading to vision loss. The development, maintenance and function of photoreceptors has been studied in a wide range of model organisms, including the zebrafish. Uniquely, the adult zebrafish is capable of regenerating the photoreceptors and other retinal cells types in response to injury. While immense work has been done to understand the function of genes involved in retinal development, regeneration, and blinding eye diseases, our understanding about many of these genes is still incomplete. The two genes studied in this dissertation have been either implicated in retinal development, degeneration, or regeneration. Calpain-5 (capn5) is a member of a family of calcium-dependent, non-lysosomal cysteine proteases. A mutation in CAPN5 has been shown to cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). ADNIV is a devastating inherited autoimmune disease of the eye that displays features commonly seen in other eye diseases, such as retinitis pigmentosa and diabetic retinopathy, and ultimately results in retinal degeneration and blindness. When activated by influxes of calcium, calpains can either degrade their protein substrates or modify the activity of their targets through proteolytic processing. Although capn5 has been extensively studied in the brain, very little is known about its role during retinal development or in the adult eye. The second gene of interest is Hairyrelated (Her) factor Her9, a member of the Hairy/Enhancer of Split (Hes) superfamily of genes. Her9 is a basic-helix-loop-helix-orange (bHLH-O) transcription factor. Her factors have been previously shown to play roles in neural tube closure, floor plate development, and development of various components of the central nervous system as well as the cranial sensory placodes. However, the role of her9 in retinal development and regeneration is still poorly understood. Chapter 1 of this dissertation is an overview of retinal development, with an emphasis on photoreceptor development, retinal degenerative diseases, regeneration, and the known roles of capn5 and her9 in other tissues. Chapter 2 characterizes the expression of capn5 during development and the adult retina. Using acute light damage and a zebrafish model for chronic rod degeneration and regeneration, this study provides evidence that capn5 has a role in photoreceptor maintenance, survival, and regeneration. Chapter 3 characterizes the expression of her9 during retinal development and uses a combination of molecular and behavioral experiments to characterize the retinal phenotypes present in a her9 CRISPR/Cas9 knockout. The data presented in this chapter demonstrate that Her9 plays a role in photoreceptor differentiation, maintenance and survival. Chapter 4 examines other phenotypes present in the her9 knockout. Craniofacial, pigment, and gut defects provide supporting evidence that Her9 is required for the differentiation and survival of neural crest cell lineages. Chapter 5 is a discussion of the conclusions we can draw from these studies, potential future directions for this work, and how these results impact our broader understanding of retinal development and regeneration

Using MapReduce Streaming for Distributed Life Simulation on the Cloud

Distributed software simulations are indispensable in the study of large-scale life models but often require the use of technically complex lower-level distributed computing frameworks, such as MPI. We propose to overcome the complexity challenge by applying the emerging MapReduce (MR) model to distributed life simulations and by running such simulations on the cloud. Technically, we design optimized MR streaming algorithms for discrete and continuous versions of Conway’s life according to a general MR streaming pattern. We chose life because it is simple enough as a testbed for MR’s applicability to a-life simulations and general enough to make our results applicable to various lattice-based a-life models. We implement and empirically evaluate our algorithms’ performance on Amazon’s Elastic MR cloud. Our experiments demonstrate that a single MR optimization technique called strip partitioning can reduce the execution time of continuous life simulations by 64%. To the best of our knowledge, we are the first to propose and evaluate MR streaming algorithms for lattice-based simulations. Our algorithms can serve as prototypes in the development of novel MR simulation algorithms for large-scale lattice-based a-life models.https://digitalcommons.chapman.edu/scs_books/1014/thumbnail.jp